RESUMEN
A thickness shear mode (TSM) quartz sensor has been used to characterize the substantivity, viscoelasticity, and mucoadhesive properties of low crystallinity cellulose (LCC) films. LCC is a novel pharmaceutical excipient that has been attributed with mucoadhesive properties. Thin films of LCC were deposited onto TSM sensors by a spin coating technique. The films were treated by passing water or 1.0% w/v mucin solution (pH 3.7 or 7.0) over the surface. Changes in the mass and viscosity of the film were observed by monitoring changes in the impedance spectra of the coated TSM sensors. Scanning electron micrographs (SEMs) of each film were used to assist the interpretation of the TSM sensor data. This study showed that LCC forms highly tenacious and viscoelastic films able to withstand prolonged (approximately 1 h) exposure to both water and mucin solution. Furthermore, these results indicate that the films may have mucoadhesive properties as LCC was found to bind significant (P<0.05) amounts of mucin in comparison with control measurements. Mucin binding to the LCC sensor was greater at pH 3.7 (P<0.05) than at pH 7.0, suggesting that the LCC formulation is mucoadhesive under these conditions.
Asunto(s)
Celulosa , Excipientes , Tecnología Farmacéutica , Química Farmacéutica , Mucinas/metabolismoRESUMEN
The genome verified C. elegans free-living nematode model is a new tool for investigating gene expression in human and animal nematode parasites. There is limited information on designating glutathione S-transferase (GST) to specific classes in lower invertebrates such as nematodes. Following cloning, amino acid sequence alignment, recombinant expression and Western blotting we provide evidence of a new GST class in nematodes or lower invertebrates.
Asunto(s)
Caenorhabditis elegans/enzimología , Glutatión Transferasa/clasificación , Nematodos/enzimología , Proteoma , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/química , Clonación Molecular , Glutatión Transferasa/química , Cinética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Parásitos/enzimología , Filogenia , Proteoma/química , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Peptide-1-[N-[2-succinamidylethyl]amino]anthraquinones containing five seven amino acid residues including the KCR motif important in AP-1 protein binding to DNA have been synthesised as potential transcription factor inhibitors. These anthraquinone-peptides showed DNA intercalative binding and inhibition of AP-1 protein binding to its DNA consensus sequence.
Asunto(s)
Antraquinonas/farmacología , Fragmentos de Péptidos/farmacología , Factor de Transcripción AP-1/antagonistas & inhibidores , Secuencias de Aminoácidos , Antraquinonas/síntesis química , Sitios de Unión , Técnicas Químicas Combinatorias , Secuencia de Consenso , ADN/metabolismo , Electroforesis , Humanos , Sustancias Intercalantes/síntesis química , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Relación Estructura-Actividad , TemperaturaRESUMEN
Piliostigma thonningii, Ocimum gratissimum, Nauclea latifolia and Alstonia boonei are used in Nigerian traditional medicines against gastrointestinal helminths of animals and man. Proanthocyanidins were detected in Piliostigma and Nauclea, but not Alstonia or Ocimum. Extracts of these plants killed 50% of brine shrimp nauplii at <10 ppm (Nauclea), 100 ppm (Piliostigma) and <1000 ppm (Ocimum and Alstonia), the Nauclea LD50 being similar to the anthelmintic drug piperazine. Extracts were also toxic to the parasitic nematode Haemonchus infective L3 stage. Nematode glutathione-S-transferases (GSTs) are potential drug targets. Apart from Alstonia all the medicinal plants contained heat-stable inhibitory activities against recombinant Ascaris and Onchocerca GSTs in vitro. Piliostigma, Ocimum and Nauclea had IC50s of 2, 10 and 15 microg/mL respectively for Ascaris GST and 4, 8, 28 microg/mL respectively for Onchocerca GST. We suggest that the inhibitory properties of some of these Nigerian plant extracts against GST may contribute to the pharmacological basis of their efficacy against helminths in traditional herbal use.
Asunto(s)
Antocianinas/farmacología , Antioxidantes/farmacología , Glutatión Transferasa/antagonistas & inhibidores , Nematodos/efectos de los fármacos , Extractos Vegetales/farmacología , Plantas Medicinales/química , Proantocianidinas , Animales , Artemia , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Glutatión Transferasa/química , Glutatión Transferasa/fisiología , Humanos , Análisis de los Mínimos Cuadrados , Modelos Lineales , Medicinas Tradicionales Africanas , Nematodos/enzimología , Nigeria , Placenta/enzimologíaRESUMEN
BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.
Asunto(s)
Sondas de ADN , ADN de Neoplasias/análisis , Colorantes Fluorescentes , Melanoma/genética , Óxidos de Nitrógeno , Antraquinonas , Proteína Quinasa CDC2/metabolismo , Ciclo Celular , Ciclina B/metabolismo , Ciclina B1 , Diagnóstico por Imagen/métodos , Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente , Expresión Génica , Proteínas Fluorescentes Verdes , Humanos , Rayos Infrarrojos , Proteínas Luminiscentes/metabolismo , Melanoma/metabolismo , Melanoma/patología , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Estructura Molecular , Quinolonas/metabolismo , Espectrofotometría/métodos , Compuestos de Tosilo/metabolismo , Células Tumorales CultivadasRESUMEN
A series of beta-carbonyl substituted glutathione conjugates were prepared and evaluated as inhibitors of OvGST2. Their specificity for the parasite derived protein was assessed through comparison with their inhibition of human piGST. Inhibition of OvGST2 has been demonstrated at low micromolar concentrations for these conjugates and selectivity for OvGST2 over human pi-GST of greater than 10-fold has been achieved.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Filaricidas/síntesis química , Glutatión Transferasa/antagonistas & inhibidores , Glutatión/análogos & derivados , Glutatión/síntesis química , Onchocerca volvulus/enzimología , Animales , Inhibidores Enzimáticos/farmacología , Filaricidas/farmacología , Glutatión/farmacología , Indicadores y Reactivos , Onchocerca volvulus/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The paper presents the first report of the purification of an invertebrate cysteine conjugate beta-lyase (CCBL). CCBL activity was shown to predominate within the cytosolic fraction of tissue from the tapeworm Moniezia expansa. The monomeric cytosolic enzyme was isolated with a M(r) of 72 kDa and co-purified with transaminase activity towards L-aspartate. The substrate profile for M. expansa CCBL is different from that of mammalian CCBLs. Exploiting the differences in mammalian and parasite substrate profiles will facilitate the development of helminth targeted conjugates which will not be activated by host (mammalian) CCBLs.
Asunto(s)
Liasas de Carbono-Azufre/aislamiento & purificación , Cestodos/enzimología , Animales , Liasas de Carbono-Azufre/química , Intestinos/parasitología , Ovinos/parasitologíaRESUMEN
Thickness shear mode (TSM) biosensors have many potential applications within the pharmaceutical sciences as a means of measuring mass changes in the nanogram range, film thickness, viscosity and shear moduli. This study addresses the possible use of the TSM sensor as a biosensor for measuring drug partition coefficients. In order to realise this potential, some fundamental understanding is required of the behaviour of lipid films on the sensor. The present study characterises the behaviour of fatty acid multilayers as a suitable model chemical system. Frequency shifts and impedance spectra are presented for multilayers of three fatty acid films coated on to the sensor using a Langmuir-Blodgett trough. The results indicate that the frequency shift is non-linear at lower numbers of fatty acid layers but the response is Sauerbrey-like at higher numbers of layers. Also at high numbers of layers, changes in the impedance spectra indicate viscoelastic behaviour in thicker membranes. An inverse relationship is observed between chain length and frequency shift, which is attributed to variations in the topography of the sensor surface. This work demonstrates the importance of fully characterising the physical behaviour of the lipid multilayers prior to using these systems for the measurement of drug partition coefficients.
Asunto(s)
Técnicas Biosensibles , Ácidos Eicosanoicos/química , Ácidos Grasos/química , Ácidos Esteáricos , Tecnología Farmacéutica/instrumentaciónRESUMEN
Living organisms employ a variety of metabolic pathways when detoxifying xenobiotic compounds, including the formation of cysteine S-conjugates via glutathione conjugation. However, cysteine conjugate beta-lyase (CCBL) catalysed beta-cleavage, of certain cysteine conjugates, is known to cause cytotoxicity. This study represents the first investigation into the expression of CCBL and other associated enzymes in helminth species. A survey of the three major groups of parasitic helminths [cestodes (Moniezia expansa), digeneans (Fasciola hepatica) and nematodes (Necator americanus, Heligmosomoides polygyrus)] has been made. The presence of CCBL enzymes within Moniezia expansa, Necator americanus and Heligmosomoides polygyrus has been established. Each species was screened for gamma-glutamyl transpeptidase activity and transaminase activity towards L-aspartate, L-alanine, L-albizziin and L-phenylalanine. Aspartate and alanine aminotransferase activity were detected in all four species tested. Gamma-glutamyl transpeptidase activity was only detected in Moniezia expansa and Necator americanus.
Asunto(s)
Liasas de Carbono-Azufre/metabolismo , Proteínas del Helminto/metabolismo , Alanina/metabolismo , Animales , Ácido Aspártico/metabolismo , Cestodos/enzimología , Fasciola hepatica/enzimología , Nematodos/enzimología , Especificidad de la Especie , Transaminasas/metabolismo , gamma-Glutamiltransferasa/metabolismoAsunto(s)
Aspartato Aminotransferasas/metabolismo , Liasas de Carbono-Azufre/metabolismo , Carboxiliasas/metabolismo , Clostridium perfringens/enzimología , Enterococcus faecalis/enzimología , Escherichia coli/enzimología , Glutamato Descarboxilasa/metabolismo , Cinética , Tirosina Descarboxilasa/metabolismoRESUMEN
A recombinant glutathione S-transferase (GST) (EC 2.5.1.18) from the parasitic nematode Ascaris suum (AsGST1) displays specific activity with a variety of model substrates and secondary products of lipid peroxidation. The AsGST1 interacts with a range of model inhibitors, haematin-related compounds, bile acids and anthelminthics. The reported variations in biochemical activity correlate with structural differences observed by homology modelling. Here, differences in the topography of the proposed substrate binding site between the AsGST1 and the host GSTs were identified. A rabbit polyclonal antiserum was raised against the glutathione-binding proteins of A. suum and specific antibodies against AsGST1 were affinity-purified using the recombinant protein. These antibodies were used to localize the AsGST1 in adult worms by immunohistochemical staining. The strongest immunostaining for AsGST1 was localized in the intestine in all worms examined. This suggests that the enzyme may be responsible for the metabolism of materials that are incorporated from the environment, as well as for molecules that are excreted or secreted from the parasite to the environment. It also demonstrates the accessibility of the enzyme to an inhibitor or blocking antibody. In addition, the structure and sequence of the gene encoding AsGST1 have been determined. Southern-blot analyses of the AsGST1 gene suggests that it is a single-copy gene. The nucleotide sequence analysis revealed that the gene is composed of four exons and three introns, and potential regulatory elements were identified in the 5' flanking sequence.
Asunto(s)
Ascaris suum/enzimología , Genes de Helminto , Glutatión Transferasa/aislamiento & purificación , Proteínas del Helminto/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/inmunología , Ascaris suum/genética , Ascaris suum/inmunología , Secuencia de Bases , Sitios de Unión , Inhibidores Enzimáticos/farmacología , Femenino , Glutatión Transferasa/antagonistas & inhibidores , Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/inmunología , Glutatión Transferasa/metabolismo , Proteínas del Helminto/antagonistas & inhibidores , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Proteínas del Helminto/metabolismo , Técnicas para Inmunoenzimas , Intestinos/enzimología , Masculino , Ratones , Modelos Moleculares , Técnicas de Sonda Molecular , Datos de Secuencia Molecular , ARN de Helminto/genética , ARN Mensajero/genética , Conejos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The C-S lysis of L-cysteine conjugates is one biotransformation pathway which is responsible for the generation of mutagenic and cytotoxic metabolic species. Thirteen cysteine S-conjugates were synthesized in our laboratories and incubated with aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) enzymes from porcine heart tissue. The C-S lyase (CSL) activity for each enzyme-substrate combination was determined. ASAT and ALAT were shown to exhibit CSL activity and it was also demonstrated that this activity was inhibited in the presence of the pyridoxal phosphate-dependent enzyme inhibitor amino(oxyacetic acid) confirming the pyridoxal phosphate-dependent mechanism by which C-S lysis is known to take place. This finding has potentially important implications for the risk assessment of compounds which produce L-cysteine conjugates during their biotransformation.
Asunto(s)
Alanina Transaminasa/metabolismo , Aspartato Aminotransferasas/metabolismo , Cisteína Sintasa/farmacocinética , Animales , Biotransformación , Cisteína/análogos & derivados , Cisteína/metabolismo , Modelos Moleculares , Miocardio/citología , Miocardio/metabolismo , Especificidad por Sustrato , PorcinosRESUMEN
One biotransformation pathway which is responsible for the generation of mutagenic and cytotoxic metabolites is that of the C-S lysis (CSL) of L-cysteine conjugates. Thirteen cysteine S-conjugates, synthesised in our laboratories, were incubated with porcine heart aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), and the C-S lyase activity for each enzyme-substrate combination was determined. ASAT and ALAT were shown to exhibit CSL activity. It was also demonstrated that this activity was inhibited in the presence of the pyridoxal phosphate (PLP)-dependent enzyme inhibitor amino(oxyacetic acid) (AOAA) confirming the pyridoxal phosphate dependent mechanism by which C-S lysis is known to take place. Since the activities of these enzymes are used as biomarkers for the assessment of organ damage, the potential interaction of L-cysteine conjugates with them may suppress their activity through direct inhibition.
Asunto(s)
Alanina Transaminasa/metabolismo , Aspartato Aminotransferasas/metabolismo , Cisteína/análogos & derivados , Cisteína/metabolismo , Animales , Biotransformación , Miocardio/enzimología , PorcinosRESUMEN
The DNA binding and cytotoxicity of four intercalating agents, namely bis-alkylamino (-N(CH2)2N(CH3)2) substituted anthraquinone, anthrapyrazole and anthracene, and mono (N(CH2)2N(CH3)2) acridinone, have been compared with their respective aliphatic amine N-oxides -N(CH2)2N+(O-)(CH3)2. The results show that, unlike the intercalators, the N-oxides do not bind to DNA. Molecular modelling illustrates that the delta + nature of the intercalator alkylamino side chains in the protonated form allows for an attractive electrostatic interaction with phosphates of the DNA backbone, whereas the delta- partial charge on the N-oxide makes such an interaction not permissible; indeed, the electrostatic interaction with the DNA phosphates will be repulsive. The N-oxides show little or no cytotoxicity against V79 cells at concentrations equimolar to the IC90 (concentration that inhibits 90% of cell proliferation) of the respective intercalators. However, the cytotoxicity of anthrapyrazole N-oxide against hypoxic V79 cells in the presence of an activating system of S9 liver fraction was enhanced significantly. The results indicate that N-oxides of DNA-affinic agents have potential as bioreductive prodrugs, since they possess low aerobic toxicity but under hypoxic conditions can be metabolised to a potent cytotoxic species presumed to be a DNA-binding tertiary amine.